Lettuce variety 45-21 rz, lorquinas rz

ABSTRACT

The present invention relates to a  Lactuca sativa  seed designated 45-21 RZ, which exhibits a combination of traits including resistance to downy mildew ( Bremia lactucae ) races Bi:1 to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid ( Nasonovia ribisnigri ) biotype Nr:0, very slow bolting, moderately to deeply incised margins of the mature leaves and a large head size. The present invention also relates to a  Lactuca sativa  plant produced by growing the 45-21 RZ seed. The invention further relates to methods for producing the lettuce cultivar, represented by lettuce variety 45-21 RZ.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSer. No. 61/579,918, filed Dec. 23, 2011.

The foregoing application, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a new lettuce (Lactuca sativa) varietywhich exhibits a combination of traits including resistance to downymildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size.

BACKGROUND OF THE INVENTION

All cultivated forms of lettuce belong to the highly polymorphicspecies, Lactuca sativa, which is grown for its edible head and leaves.As a crop, lettuces are grown commercially wherever environmentalconditions permit the production of an economically viable yield.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, scorzonera,dandelion, artichoke and chrysanthemum. Sativa is one of about 300species in the genus Lactuca.

Lettuce cultivars are susceptible to a number of pests and diseases suchas downy mildew (Bremia lactucae). Every year this disease leads tomillions of dollars of lost lettuce crop throughout the world. Downymildew (Bremia lactucae) is highly destructive on lettuce grown atrelatively low temperature and high humidity. Downy mildew is caused bya fungus, Bremia lactucae, which can be one of the following strains:NL1, NL2, NL4, NL5, NL6, NL7, NL10, NL12, NL13, NL14, NL15, NL16, Bl:17,Bl:18, Bl:20, Bl:21, Bl:22, Bl:23, Bl:24, Bl:25, Bl:26, Bl:27, Bl:28(Van Ettekoven, K. et al., “Identification and denomination of ‘new’races of Bremia lactucae,” In: Lebeda, A. and Kristkova, E (eds.),Eucarpia Leafy Vegetables, 1999, Palacky University, Olomouc, CzechRepublic, pp. 171-175; Van der Arend et al. “Identification anddenomination of “new” races of Bremia lactucae in Europe by IBEB until2002.” In: Van Hintum, Th et al. (eds.), Eucarpia Leafy VegetablesConference 2003, Centre for Genetic Resources, Wageningen, TheNetherlands, p. 151; Plantum NL (Dutch association for breeding, tissueculture, production and trade of seeds and young plants), Van der Arendet al. “Identification and denomination of “new” races of Bremialactucae in Europe by IBEB until 2002.” In: Van Hintum, Th et al.(eds.), Eucarpia Leafy Vegetables Conference 2003, Centre for GeneticResources, Wageningen, The Netherlands, p. 151; Plantum NL (Dutchassociation for breeding, tissue culture, production and trade of seedsand young plants); IBEB press release “New race of Bremia lactucae Bl:27identified and nominated”, May 2010; Plantum NL (Dutch association forbreeding, tissue culture, production and trade of seeds and youngplants), “New race of Bremia lactucae Bl:28 identified and nominated”,March 2011), Ca-I, Ca-IIA, Ca-IIB, Ca-III, Ca-IV (Schettini, T. M.,Legg, E. J., Michelmore, R. W., 1991. Insensitivity to metalaxyl inCalifornia populations of Bremia lactucae and resistance of Californialettuce cultivars to downy mildew. Phytopathology 81(1). p. 64-70), andCa-V, Ca-VI, Ca-VII, Ca-VIII (Michelmore R. & Ochoa. O. “BreedingCrisphead Lettuce.” In: California Lettuce Research Board, Annual Report2005-2006, 2006, Salinas, California, pp. 55-68).

Downy mildew causes pale, angular, yellow areas bounded by veins on theupper leaf surfaces. Sporulation occurs on the opposite surface of theleaves. The lesions eventually turn brown, and they may enlarge andcoalesce. These symptoms typically occur first on the lower leaves ofthe lettuce, but under ideal conditions may move into the upper leavesof the head. When the fungus progresses to this degree, the head cannotbe harvested. Less severe damage requires the removal of more leavesthan usual, especially when the lettuce reaches its final destination.

Of the various species of aphids that feed on lettuce, thecurrant-lettuce aphid (Nasonovia ribisnigri) is the most destructivespecies because it feeds both on the leaves of the lettuce as well asdeep in the heart of the lettuce, making it difficult to control withconventional insecticides. The lettuce aphid feeds by sucking sap fromthe lettuce leaves. Although direct damage to the lettuce may belimited, its infestation has serious consequences because the presenceof aphids makes lettuce unacceptable to consumers.

Next to diseases and pests such as downy mildew and the currant-lettuceaphid, bolting is considered a serious problem in lettuce production,because it limits the growing cycle of a lettuce head, and thereforereduces yield. When bolting starts bitter components are produced in theleaves which results in a disappointing taste experience for consumers.Additionally the plant's stem is elongating and causes deformation ofthe head. Such heads cannot be properly packed, are not accepted byconsumers and are therefore not marketable.

Yield is an important factor for a profitable production of iceberglettuce. Yield is based on the number of harvested heads per acre andtheir head weight. Especially when iceberg lettuce is used forprocessing and sold as a pre-cut, pre-washed product, higher yields arethe driving factor to reduce raw product cost. High head weights canonly be reached when the heads are large sized with sufficient volume.Smaller-sized, very dense heads are more prone to tipburn problems. Alarge head size also provides sufficient space inside the head forfurther growing. Especially when supply of lettuce is high due tofavorable weather conditions, large-headed lettuce varieties providemore flexibility to delay the harvest moment.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a need, therefore, for an iceberg lettuce variety whichexhibits a combination of traits including resistance against downymildew (Bremia lactucae) races Bl:1-28 and CA-I to CA-VIII, resistanceagainst currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, veryslow bolting, and a large head size.

The present invention addresses this need by providing a new type oficeberg lettuce (Lactuca sativa) variety, designated 45-21 RZ andalternatively designated “Lorquinas RZ”. Lettuce cultivar 45-21 RZ orLoquinas RZ may exhibit a combination of traits including resistance todowny mildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size.

The present invention provides seeds of lettuce cultivar 45-21 RZ, whichhave been deposited with the National Collections of Industrial, Marineand Food Bacteria (NCIMB) in Bucksburn, Aberdeen AB21 9YA, Scotland, UKand have been assigned NCIMB Accession No. 41901.

In one embodiment, the invention provides a lettuce plant which mayexhibit a combination of traits including resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size, representative seed of which have been deposited underNCIMB Accession No. 41901.

In one embodiment, the invention provides a lettuce plant which mayexhibit a combination of traits including resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves, alarge head size and moderate to slight blistering, representative seedof which have been deposited under NCIMB Accession No. 41901.

In one embodiment, the invention provides a lettuce plant designated45-21 RZ, representative seed of which have been deposited under NCIMBAccession No. 41901.

In an embodiment of the present invention, there also is provided partsof a lettuce plant of the invention, which may include parts of alettuce plant exhibiting a combination of traits including resistance todowny mildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size, or parts of a lettuce plant having any ofthe aforementioned resistance(s) and a combination of traits includingone or more morphological or physiological characteristics tabulatedherein, including parts of lettuce variety 45-21 RZ, wherein the plantparts are involved in sexual reproduction, which include, withoutlimitation, microspores, pollen, ovaries, ovules, embryo sacs or eggcells and/or wherein the plant parts are suitable for vegetativereproduction, which include, without limitation, cuttings, roots, stems,cells or protoplasts and/or wherein the plant parts are tissue cultureof regenerable cells in which the cells or protoplasts of the tissueculture are derived from a tissue such as, for example and withoutlimitation, leaves, pollen, embryos, cotyledon, hypocotyls, meristematiccells, roots, root tips, anthers, flowers, seeds or stems. The plants ofthe invention from which such parts may come from include those whereinrepresentative seed of which has been deposited under NCIMB AccessionNo. 41901 or lettuce variety or cultivar designated 45-21 RZ, as well asseed from such a plant, plant parts of such a plant (such as thosementioned herein) and plants from such seed and/or progeny of such aplant, advantageously progeny exhibiting such combination of suchtraits, each of which, is within the scope of the invention; and suchcombination of traits.

In another embodiment there is a plant grown from seeds, representativeseed of which having been deposited under NCIMB Accession No. 41901.

In a further embodiment there is a plant regenerated from theabove-described plant parts or regenerated from the above-describedtissue culture. Advantageously such a plant may have morphologicaland/or physiological characteristics of lettuce variety 45-21 RZ and/orof a plant grown from seed, representative seed of which having beendeposited under NCIMB Accession No. NCIMB 41901—including withoutlimitation such plants having all of the morphological and physiologicalcharacteristics of lettuce variety 45-21 RZ and/or of plant grown fromseed, representative seed of which having been deposited under NCIMBAccession No. NCIMB 41901. Advantageously, such a plant demonstrates thetraits of resistance against downy mildew (Bremia lactucae) racesBl:1-28 and CA-I to CA-VIII, resistance against currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size.

Accordingly, in still a further embodiment, there is provided a lettuceplant having all of the morphological and physiological characteristicsof lettuce variety 45-21 RZ, representative seed of which having beendeposited under NCIMB Accession No. 41901. Such a plant may be grownfrom the seeds, regenerated from the above-described plant parts, orregenerated from the above-described tissue culture. A lettuce planthaving any of the aforementioned resistance(s), and one or moremorphological or physiological characteristics recited or tabulatedherein, and a lettuce plant advantageously having all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred. Parts of such plants—such as those plant partsabove-mentioned—are encompassed by the invention.

In one embodiment, there is provided progeny of lettuce cultivar 45-21RZ produced by sexual or vegetative reproduction, grown from seeds,regenerated from the above-described plant parts, or regenerated fromthe above-described tissue culture of the lettuce cultivar or a progenyplant thereof, representative seed of which having been deposited underNCIMB Accession No. 41901. The progeny may have any of theaforementioned resistance(s), and one or more morphological orphysiological characteristics recited or tabulated herein, and a progenyplant advantageously having all of the aforementioned resistances andthe characteristics recited and tabulated herein, is preferred.Advantageously, the progeny demonstrate the traits of resistance againstdowny mildew (Bremia lactucae) races Bl:1-28 and CA-I to CA-VIII,resistance against currant-lettuce aphid (Nasonovia ribisnigri) biotypeNr:0, very slow bolting, moderately to deeply incised margins of themature leaves and a large head size.

Progeny of the lettuce variety 45-21 RZ may be modified in one or moreother characteristics, in which the modification is a result of, forexample and without limitation, mutagenesis or transformation with atransgene.

In still another embodiment, the present invention provides progeny oflettuce cultivar 45-21 RZ produced by sexual or vegetative reproduction,grown from seeds, regenerated from the above-described plant parts, orregenerated from the above-described tissue culture of the lettucecultivar or a progeny plant thereof, in which the regenerated plantshows a combination of traits including resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size.

In still a further embodiment, the invention may comprise a method ofproducing a hybrid lettuce seed which may comprise crossing a firstparent lettuce plant with a second parent lettuce plant and harvestingthe resultant hybrid lettuce seed, in which the first parent lettuceplant or the second parent lettuce plant may be a lettuce plant of theinvention, e.g. a lettuce plant having a combination of traits includingresistance to downy mildew (Bremia lactucae) races Bl:1 to Bl:28 andCA-I to CA-VIII, resistance to currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0, very slow bolting, moderately to deeplyincised margins of the mature leaves and a large head size and one ormore morphological or physiological characteristics tabulated herein,including a lettuce plant of lettuce cultivar 45-21 RZ, representativeseed of which having been deposited under NCIMB 41901.

In another embodiment, the invention may comprise producing a lettuceplant having a combination of traits including resistance to downymildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size comprising: crossing a mother lettuce plantwith a father lettuce plant to produce a hybrid seed; growing saidhybrid seed to produce a hybrid plant; selfing said hybrid plant toproduce F2 progeny seed; growing said F2 progeny seed to produceF2-plants; selecting said F2-plants for exhibiting a combination oftraits including resistance to downy mildew (Bremia lactucae) races Bl:1to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size.

Advantageously the selfing and selection may be repeated; for example atleast once, or at least twice, thrice, four times, five times, six timesor more, to produce F3 or F4 or F5 or F6 or subsequent progeny,especially as progeny from F2 may exhibit the aforementioned combinationof traits, and may be desirable.

In still a further embodiment, the invention may comprise a method ofproducing a lettuce cultivar containing a combination of traitsincluding resistance to downy mildew (Bremia lactucae) races Bl:1 toBl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size.

The invention even further relates to a method of producing lettucewhich may comprise: (a) cultivating to the vegetative plant stage aplant of lettuce variety 45-21 RZ, representative seed of which havingbeen deposited under NCIMB Accession No. NCIMB 41901, and (b) harvestinglettuce leaves or heads from the plant. The invention furthercomprehends packaging the lettuce plants, heads or leaves.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, and “comprising” and the like(e.g., “includes”, “included”, “including”, “contains”, “contained”,“containing”, “has”, “had”, “having”, etc.) can have the meaningascribed to them in US Patent law, i.e., they are open ended terms. Forexample, any method that “comprises,” “has” or “includes” one or moresteps is not limited to possessing only those one or more steps and alsocovers other unlisted steps. Similarly, any plant that “comprises,”“has” or “includes” one or more traits is not limited to possessing onlythose one or more traits and covers other unlisted traits. Similarly,the terms “consists essentially of” and “consisting essentially of” havethe meaning ascribed to them in US Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. See also MPEP §2111.03. In addition, the term “about” is usedto indicate that a value includes the standard deviation of error forthe device or method being employed to determine the value.

These and other embodiments are disclosed or are obvious from andencompassed by the following Detailed Description.

Deposit

The Deposit with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on Nov. 22, 2011, under depositaccession number NCIMB 41901 was made pursuant to the terms of theBudapest Treaty. Upon issuance of a patent, all restrictions upon thedeposit will be removed, and the deposit is intended to meet therequirements of 37 CFR §1.801-1.809. The deposit will be maintained inthe depository for a period of 30 years, or 5 years after the lastrequest, or for the effective life of the patent, whichever is longer,and will be replaced if necessary during that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawing, in which:

FIG. 1 is an illustration of six different shapes of the fourth leaffrom a 20-day old seedling grown under optimal conditions.

FIG. 2 is a picture of the shape of the fourth leaf of 45-21 RZ.

FIG. 3 is a picture of the shape of a mature leaf of 45-21 RZ.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of a new lettuce variety herein referred to aslettuce variety 45-21 RZ. Lettuce variety 45-21 RZ is a uniform andstable line, distinct from other such lines.

In a preferred embodiment, the specific type of breeding method employedfor developing a lettuce cultivar is pedigree selection, where bothsingle plant selection and mass selection practices are employed.Pedigree selection, also known as the “Vilmorin system of selection,” isdescribed in Fehr, W., Principles of Cultivar Development, Volume I,MacMillan Publishing Co., which is hereby incorporated by reference.

When pedigree selection is applied, in general selection is firstpracticed among F₂ plants. In the next season, the most desirable F₃lines are first identified, and then desirable F₃ plants within eachline are selected. The following season and in all subsequentgenerations of inbreeding, the most desirable families are identifiedfirst, then desirable lines within the selected families are chosen, andfinally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny from the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring.Lettuce is an obligate self-pollination species, which means that pollenis shed before stigma emergence, assuring 100% self-fertilization.Therefore, in order to optimize crossing, a method of misting may beused to wash the pollen off prior to fertilization to assure crossing orhybridization.

Parental varieties are selected from commercial varieties thatindividually exhibit one or more desired phenotypes. Additionally, anybreeding method involving selection of plants for the desired phenotypemay be used in the method of the present invention.

The F₁ may be self-pollinated to produce a segregating F₂ generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F₃, F₄, F₅, etc.) until the traits arehomozygous or fixed within a breeding population.

Lettuce variety 45-21 RZ was developed by crossing line “560871” and“32625” under glasshouse conditions in Fijnaart, The Netherlands in2006. The F1 resulting from this cross was grown for seed production inFijnaart, The Netherlands in 2007 under glasshouse conditions. An F2plant was selected in plastic tunnels in Daylesford, Australia, in 2008,after which F3 seed was obtained through self-pollination. F3 plantswere selected in 2009 again under field conditions in Fijnaart, TheNetherlands where also F4 seed production in took place. Subsequently,an F4 plant was selected out of the F4 seeds in field conditions inMurcia, Spain in 2009. Finally, a uniform F5 line was established. Thisline was multiplied in 2010 under plastic tunnel conditions inDaylesford, Australia having seed lot number 10R.535, seeds of whichwere deposited under Accession No. NCIMB 41901 on Nov. 22, 2011.

In one embodiment, a plant of the invention has all the morphologicaland physiological characteristics of lettuce variety 45-21 RZ. Thesecharacteristics of a lettuce plant of the invention, e.g. variety 45-21RZ, are summarized in Tables 1 and 2.

Next to the morphological and physiological characteristics mentioned inTables 1 and 2, a plant of the invention also exhibits resistance todowny mildew (Bremia lactucae) and currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0.

As used herein resistance against Bremia lactucae Regel. is defined asthe ability of a plant to resist infection by each of the variousstrains Bl:1-28, Ca-V to Ca-VIII of Bremia lactucae Regel. in all stagesbetween the seedling stage and the harvestable plant stage. Bl:1-28means strains NL1, NL2, NL4, NL5, NL6, NL7, NL10, NL12, NL13, NL14,NL15, NL16, Bl:17, Bl:18, Bl:20, Bl:21, Bl:22, Bl:23, Bl:24, Bl:25,Bl:26, Bl:27, Bl:28 (Van Ettekoven K, Van der Arend A J M, 1999.identification and denomination of ‘new’ races of Bremia lactucae. In:Lebeda A, Kristkova E (eds.) Eucarpia leafy vegetables '99. PalackyUniversity, Olomouc, Czech Republic, 1999: 171-175; Van der Arend, A. J.M., Gautier, J., Guenard, M., Michel, H., Moreau, B., de Ruijter, J.,Schut, J. W. and de Witte, I. (2003). Identification and denomination of‘new’ races of Bremia lactucae in Europe by IBEB until 2002. In:Eucarpia leafy vegetables 2003. Proceedings of the Eucarpia Meeting onleafy vegetables genetics and breeding. Noorwijkerhout, The Netherlands.Eds. Van Hintum T., Lebeda A., Pink D., Schut J. pp 151-160; Van derArend A J M, Gautier J, Grimault V, Kraan P, Van der Laan R, Mazet J,Michel H, Schut J W, Smilde D, De Witte I (2006) Identification anddenomination of “new” races of Bremia lactucae in Europe by IBEB until2006; incorporated herein by reference; Plantum N L (Dutch associationfor breeding, tissue culture, production and trade of seeds and youngplants), IBEB press release, “New race of Bremia lactucae Bl:27identified and nominated”, May 2010; Plantum N L (Dutch association forbreeding, tissue culture, production and trade of seeds and youngplants), IBEB press release, “New race of Bremia lactucae Bl:28identified and nominated”, March 2011). Ca-I to Ca-VIII means Ca-I,Ca-IIA, Ca-IIB, Ca-III, Ca-IV (Schettini, T. M., Legg, E. J.,Michelmore, R. W., 1991. Insensitivity to metalaxyl in Californiapopulations of Bremia lactucae and resistance of California lettucecultivars to downy mildew, Phytopathology 81(1). p. 64-'70), and Ca-V,Ca-VI, Ca-VII, Ca-VIII (Michelmore R. & Ochoa. 0. “Breeding CrispheadLettuce.” In: California Lettuce Research Board, Annual Report2005-2006, 2006, Salinas, California, pp. 55-68).

Resistance typically is tested by two interchangeable methods, describedby Bonnier, F. J. M. et al. (Euphytica, 61(3):203-211, 1992;incorporated herein by reference). One method involves inoculating 7-dayold seedlings, and observing sporulation 10 to 14 days later. The othermethod involves inoculating leaf discs with a diameter of 18 mm obtainedfrom a non-senescent, fully grown true leaf and observing sporulation 10days later.

As used herein, resistance against Nasonovia ribisnigri (Mosley), orcurrant-lettuce aphid, is defined as the plant characteristic whichresults in a non-feeding response of the aphid on the leaves of theplant in all stages between 5 true-leaf stage and harvestable plantstage (U.S. Pat. No. 5,977,443 to Jansen, J. P. A., “Aphid Resistance inComposites,” p. 12, 1999; incorporated herein by reference).

Resistance is tested by spreading at least ten aphids of biotype Nr:0 ona plant in a plant stage between 5 true leaves and harvestable stage,and observing the density of the aphid population on the plant as wellas the growth reduction after 14 days in a greenhouse, with temperaturesettings of 23 degrees Celsius in daytime and 21 degrees Celsius atnight. Daylength is kept at 18 hours by assimilation lights.

As used herein, a large-sized head is a mature, regular-shaped headwhich is larger in diameter than a head of the standard variety Elenas,both grown under long-day outdoor conditions at a latitude of 52°, i.e.sowing 100 days before the longest day, in an Oceanic climate(Köppen-classification: Cfb; McKnight & Hess, 2000. Physical Geography:A Landscape Appreciation. Upper Saddle River, N.J.: Prentice Hall).

As used herein, slow bolting is bolting speed, which is comparable tothe variety Salinas. As used herein, bolting is measured as the numberof days from first water date to seed stalk emergence. Bolting ismeasured from plants that were grown under long-day outdoor conditionsat a latitude of 52°, i.e. sowing 100 days before the longest day, in anOceanic climate (Köppen-classification: Cfb; McKnight & Hess, 2000.Physical Geography: A Landscape Appreciation. Upper Saddle River, N.J.:Prentice Hall). An analysis of variance with location and variety asmain effects is performed and followed by an F-test to test for asignificant varietal difference. Bolting class is also related tobolting. As used herein, bolting class is based on a scale from 1 to 5where 1=very slow, 2=slow, 3=medium, 4=rapid, 5=very rapid. As usedherein, the characteristic of bolting is by comparison to standardvarieties. As used herein, very slow bolting is a bolting speed, whichis about four days slower bolting than the variety Elenas.

As used herein, moderately to deeply incised margins of the matureleaves refers to an incision depth of the mature leaves which is deeperthan standard variety Salinas or Elenas, but less deep than that ofvariety Great Lakes 659, when cultivated under the same circumstances.For the measurement of this characteristic the deepest incision of theleaf is used.

As used herein, leaf blistering in lettuce is the amount of bubbling orwrinkling of the leaves. Among lettuce varieties different levels ofblistering may be observed. A detailed overview of the differentcategories of blistering recognized in lettuce is given in theNaktuinbouw calibration book for lettuce (2010, version 1, page 50-51),which may be obtained via e-mail: kalibratieboek@naktuinbouw.nl. Thebook is based on CPVO protocol CPVO-TP/13/4.

Embodiments of the inventions advantageously have one or more, and mostadvantageously all, of these characteristics.

In Table 1, the seed color, cotyledon shape and characteristics of thefourth leaf of “45-21 RZ” is compared with “45-32 RZ”, “45-89 RZ”,“Elenas” and “Salinas”.

TABLE 1 Character “45-21 RZ” “45-32 RZ” “45-89 RZ” “Elenas” “Salinas”Plant Type Salinas Salinas Salinas Salinas Salinas group group groupgroup group Seed Color Black (grey Black (grey Black (grey Black (greyBlack (grey brown) brown) brown) brown) brown) Cotyledon ShapeIntermediate Intermediate Intermediate Intermediate Intermediate tobroad Shape of Fourth Elongated Elongated Elongated Elongated ElongatedLeaf Rolling of Fourth Present Present Present Present Present LeafCupping of Uncupped Uncupped Uncupped Uncupped Uncupped Fourth LeafFourth Leaf Entire Entire Entire Entire Entire Apical Margin Fourth LeafModerately Coarsely Moderately Moderately Coarsley Basal Margin dentatedentate dentate dentate dentate Undulation Flat Flat Flat to slight FlatSlight to flat Green color Medium Medium to Medium to Dark to Dark togreen dark green dark green medium medium green green Anthocyanin AbsentAbsent Absent Absent Absent distribution

In Table 2, the mature leaf and head characteristics of “45-21 RZ” iscompared with 45-32 RZ, 45-89 RZ, “Elenas” and “Salinas”.

TABLE 2 Character “45-21 RZ” “45-32 RZ” “45-89 RZ” “Elenas” “Salinas”Leaf Color Medium Medium Dark green Medium Medium to green (slightlygreen (greyish) green dark green yellowish) (greyish) (greyish)(greyish) Anthocyanin Absent Absent Absent Absent Absent DistributionMargin Moderate to Moderate Moderate Moderate Moderate Incision deepDepth Margin Shallowly Shallowly Shallowly Shallowly ShallowlyIndentation dentate dentate dentate dentate dentate Undulations ModerateModerate Moderate Moderate Moderate of the Apical Margin Leaf SizeMedium to Medium to Medium Medium Medium to large large large Leaf DullDull Dull Dull Moderate to Glossiness dull Leaf Moderate to Moderate toModerate to Slight to Slight to Blistering slight slight slight moderatemoderate Leaf Intermediate Intermediate Thick Intermediate IntermediateThickness to thick to thick to thick Trichomes Absent Absent AbsentAbsent Absent Head Shape Spherical Spherical Spherical SphericalSpherical Butt Shape Rounded Rounded Rounded Rounded Rounded MidribModerately Moderately Moderately Moderately Moderately raised raisedraised raised raised

In an embodiment, the invention relates to lettuce plants that has allthe morphological and physiological characteristics of the invention andhave acquired said characteristics by introduction of the geneticinformation that is responsible for the characteristics from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cisgenesis or transgenesis. Cisgenesis is geneticmodification of plants with a natural gene, coding for an (agricultural)trait, from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or a synthetic gene.

Just as useful traits that may be introduced by backcrossing, usefultraits may be introduced directly into the plant of the invention, beinga plant of lettuce variety 45-21 RZ, by genetic transformationtechniques; and, such plants of lettuce variety 45-21 RZ that haveadditional genetic information introduced into the genome or thatexpress additional traits by having the DNA coding there for introducedinto the genome via transformation techniques, are within the ambit ofthe invention, as well as uses of such plants, and the making of suchplants.

Genetic transformation may therefore be used to insert a selectedtransgene into the plant of the invention, being a plant of lettucevariety 45-21 RZ or may, alternatively, be used for the preparation oftransgenes which may be introduced by backcrossing. Methods for thetransformation of plants, including lettuce, are well known to those ofskill in the art.

Vectors used for the transformation of lettuce cells are not limited solong as the vector may express an inserted DNA in the cells. Forexample, vectors which may comprise promoters for constitutive geneexpression in lettuce cells (e.g., cauliflower mosaic virus 35Spromoter) and promoters inducible by exogenous stimuli may be used.Examples of suitable vectors include pBI binary vector. The “lettucecell” into which the vector is to be introduced includes various formsof lettuce cells, such as cultured cell suspensions, protoplasts, leafsections, and callus. A vector may be introduced into lettuce cells byknown methods, such as the polyethylene glycol method, polycationmethod, electroporation, Agrobacterium-mediated transfer, particlebombardment and direct DNA uptake by protoplasts. To effecttransformation by electroporation, one may employ either friabletissues, such as a suspension culture of cells or embryogenic callus oralternatively one may transform immature embryos or other organizedtissue directly. In this technique, one would partially degrade the cellwalls of the chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wound tissues in a controlled manner.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate. An illustrativeembodiment of a method for delivering DNA into plant cells byacceleration is the Biolistics Particle Delivery System, which may beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a surface covered with targetlettuce cells. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge. Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually any plant species, including a plantof lettuce variety 45-21 RZ.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA may be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast.Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Moreover, advances in vectors for Agrobacterium-mediated gene transferhave improved the arrangement of genes and restriction sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. The vectors have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes.Additionally, Agrobacterium containing both armed and disarmed Ti genesmay be used for transformation. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene locustransfer. The use of Agrobacterium-mediated plant integrating vectors tointroduce DNA into plant cells, including lettuce plant cells, is wellknown in the art (See, e.g., U.S. Pats. No. 7,250,560 and 5,563,055).

Transformation of plant protoplasts also may be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, a tandemly duplicated version of the CaMV 35Spromoter, the enhanced 35S promoter (P-e35S), the nopaline synthasepromoter, the octopine synthase promoter, the figwort mosaic virus(P-FMV) promoter (see U.S. Pat. No. 5,378,619), an enhanced version ofthe FMV promoter (P-eFMV) where the promoter sequence of P-FMV isduplicated in tandem, the cauliflower mosaic virus 19S promoter, asugarcane bacilliform virus promoter, a commelina yellow mottle viruspromoter, the promoter for the thylakoid membrane proteins from lettuce(psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS) (see U.S. Pat. No.7,161,061), the CAB-1 promoter from lettuce (see U.S. Pat. No.7,663,027), the promoter from maize prolamin seed storage protein (seeU.S. Pat. No. 7,119,255), and other plant DNA virus promoters known toexpress in plant cells. A variety of plant gene promoters that areregulated in response to environmental, hormonal, chemical, and/ordevelopmental signals may be used for expression of an operably linkedgene in plant cells, including promoters regulated by (1) heat, (2)light (e.g., pea rbcS-3A promoter, maize rbcS promoter, or chlorophylla/b-binding protein promoter), (3) hormones, such as abscisic acid, (4)wounding (e.g., wunl, or (5) chemicals such as methyl jasmonate,salicylic acid, or Safener. It may also be advantageous to employorgan-specific promoters.

Exemplary nucleic acids which may be introduced to the lettuce varietyof this invention include, for example, DNA sequences or genes fromanother species, or even genes or sequences which originate with or arepresent in lettuce species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA may include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a plant of lettuce variety 45-21 RZ.Non-limiting examples of particular genes and corresponding phenotypesone may choose to introduce into a lettuce plant include one or moregenes for insect tolerance, pest tolerance such as genes for fungaldisease control, herbicide tolerance, and genes for quality improvementssuch as yield, nutritional enhancements, environmental or stresstolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s).

Alternatively, the DNA coding sequences may affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms. The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product. Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention. (See also U.S. Pat. No.7,576,262, “Modified gene-silencing RNA and uses thereof.”)

U.S. Pats. Nos. 7,230,158, 7,122,720, 7,081,363, 6,734,341, 6,503,732,6,392,121, 6,087,560, 5,981,181, 5,977,060, 5,608,146, 5,516,667, eachof which, and all documents cited therein are hereby incorporated hereinby reference, consistent with the above INCORPORATION BY REFERENCEsection, are additionally cited as examples of U.S. Patents that mayconcern transformed lettuce and/or methods of transforming lettuce orlettuce plant cells, and techniques from these US Patents, as well aspromoters, vectors, etc., may be employed in the practice of thisinvention to introduce exogenous nucleic acid sequence(s) into a plantof lettuce variety 45-21 RZ (or cells thereof), and exemplify someexogenous nucleic acid sequence(s) which may be introduced into a plantof lettuce variety 45-21 RZ (or cells thereof) of the invention, as wellas techniques, promoters, vectors etc., to thereby obtain further plantsof lettuce variety 45-21 RZ, plant parts and cells, seeds, otherpropagation material harvestable parts of these plants, etc. of theinvention, e.g. tissue culture, including a cell or protoplast, such asan embryo, meristem, cotyledon, pollen, leaf, anther, root, root tip,pistil, flower, seed or stalk.

The invention further relates to propagation material for producingplants of the invention. Such propagation material may comprise interalia seeds of the claimed plant and parts of the plant that are involvedin sexual reproduction. Such parts are for example selected from thegroup consisting of seeds, microspores, pollen, ovaries, ovules, embryosacs and egg cells. In addition, the invention relates to propagationmaterial which may comprise parts of the plant that are suitable forvegetative reproduction, for example cuttings, roots, stems, cells,protoplasts.

According to a further aspect thereof the propagation material of theinvention may comprise a tissue culture of the claimed plant. The tissueculture may comprise regenerable cells. Such tissue culture may bederived from leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.(See generally U.S. Pat. No. 7,041,876 on lettuce being recognized as aplant that may be regenerated from cultured cells or tissue).

Also, the invention comprehends methods for producing a seed of a “45-21RZ”-derived lettuce plant which may comprise (a) crossing a plant oflettuce variety 45-21 RZ, representative seed of which having beendeposited under NCIMB Accession No. NCIMB 41901, with a second lettuceplant, and (b) whereby seed of a “45-21 RZ”-derived lettuce plant forms.Such a method may further comprise (c) crossing a plant grown from“45-21 RZ”-derived lettuce seed with itself or with a second lettuceplant to yield additional “45-21 RZ”-derived lettuce seed, (d) growingthe additional “45-21 RZ”-derived lettuce seed of step (c) to yieldadditional “45-21 RZ”-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further “45-21 RZ”-derived lettuce plants.

The invention further relates to the above methods that further compriseselecting at steps b), d), and e), a 45-21 RZ-derived lettuce plant,exhibiting a combination of traits including resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size.

In particular, the invention relates to methods for producing a seed ofa “45-21 RZ”-derived lettuce plant which may comprise (a) crossing aplant of lettuce variety 45-21 RZ, representative seed of which havingbeen deposited under NCIMB Accession No. NCIMB 41901, with a secondlettuce plant and (b) whereby seed of a 45-21 RZ-derived lettuce plantforms, wherein such a method may further comprise (c) crossing a plantgrown from 45-21 RZ-derived lettuce seed with itself or with a secondlettuce plant to yield additional 45-21 RZ-derived lettuce seed, (d)growing the additional 45-21 RZ-derived lettuce seed of step (c) toyield additional 45-21 RZ-derived lettuce plants and selecting plantsexhibiting a combination of traits including resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size, and (e) repeating the crossing and growing of steps (c)and (d) for an additional 3-10 generations to further generate 45-21RZ-derived lettuce plants that exhibit a combination of traits includingresistance to downy mildew (Bremia lactucae) races Bl:1 to Bl:28 andCA-I to CA-VIII, resistance to currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0, very slow bolting, moderately to deeplyincised margins of the mature leaves and a large head size.

The invention additionally provides a method of introducing a desiredtrait into a plant of lettuce variety 45-21 RZ which may comprise: (a)crossing a plant of lettuce variety 45-21 RZ, representative seed ofwhich having been deposited under NCIMB Accession No. NCIMB 41901, witha second lettuce plant that may comprise a desired trait to produce F1progeny; (b) selecting an F1 progeny that may comprise the desiredtrait; (c) crossing the selected F1 progeny with a plant of lettucevariety 45-21 RZ, to produce backcross progeny; (d) selecting backcrossprogeny which may comprise the desired trait and the physiological andmorphological characteristic of a plant of lettuce variety 45-21 RZ;and, optionally, (e) repeating steps (c) and (d) one or more times insuccession to produce selected fourth or higher backcross progeny thatcomprise the desired trait and all of the physiological andmorphological characteristics of a plant of lettuce variety 45-21 RZ,when grown in the same environmental conditions. The invention, ofcourse, includes a lettuce plant produced by this method.

Backcrossing may also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This may be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred. When a plant of lettuce variety 45-21 RZ,representative seed of which having been deposited under NCIMB AccessionNo. NCIMB 41901, is used in backcrossing, offspring retaining thecombination of traits including resistance to downy mildew (Bremialactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size are progeny within the ambit of the invention.Backcrossing methods may be used with the present invention to improveor introduce a characteristic into a plant of the invention, being aplant of lettuce variety 45-21 RZ. See, e.g., U.S. Pat. No. 7,705,206(incorporated herein by reference consistent with the aboveINCORPORATION BY REFERENCE section), for a general discussion relatingto backcrossing.

The invention further involves a method of determining the genotype of aplant of lettuce variety 45-21 RZ, representative seed of which has beendeposited under NCIMB Accession No. NCIMB 41901, or a first generationprogeny thereof, which may comprise obtaining a sample of nucleic acidsfrom said plant and detecting in said nucleic acids a plurality ofpolymorphisms. This method may additionally comprise the step of storingthe results of detecting the plurality of polymorphisms on a computerreadable medium. The plurality of polymorphisms are indicative of and/orgive rise to the expression of the morphological and physiologicalcharacteristics of lettuce variety 45-21 RZ.

There are various ways of obtaining genotype data from a nucleic acidsample. Genotype data may be gathered which is specific for certainphenotypic traits (e.g. gene sequences), but also patterns of randomgenetic variation may be obtained to construct a so-called DNAfingerprint. Depending on the technique used a fingerprint may beobtained that is unique for lettuce variety 45-21 RZ. Obtaining a uniqueDNA fingerprint depends on the genetic variation present in a varietyand the sensitivity of the fingerprinting technique. A technique knownin the art to provide a good fingerprint profile is called AFLPfingerprinting technique (See generally U.S. Pat. No. 5,874,215), butthere are many other marker based techniques, such as RFLP (orRestriction fragment length polymorphism), SSLP (or Simple sequencelength polymorphism), RAPD (or Random amplification of polymorphic DNA)VNTR (or Variable number tandem repeat), Microsatellite polymorphism,SSR (or Simple sequence repeat), STR (or Short tandem repeat), SFP (orSingle feature polymorphism) DArT (or Diversity Arrays Technology), RADmarkers (or Restriction site associated DNA markers) (e.g. Baird et al.PloS One Vol. 3 e3376, 2008; Semagn et al. African Journal ofBiotechnology Vol. 5 number 25 pp. 2540-2568, 29 December, 2006).Nowadays, sequence-based methods are utilizing Single NucleotidePolymorphisms (SNPs) that are randomly distributed across genomes, as acommon tool for genotyping (e.g. Elshire et al. PloS One Vol. 6: e19379,2011; Poland et al. PloS One Vol. 7: e32253; Truong et al. PLoS One Vol.7 number 5: e37565, 2012).

With any of the aforementioned genotyping techniques, polymorphisms maybe detected when the genotype and/or sequence of the plant of interestis compared to the genotype and/or sequence of one or more referenceplants. As used herein, the genotype and/or sequence of a referenceplant may be derived from, but is not limited to, any one of thefollowing: parental lines, closely related plant varieties or species,complete genome sequence of a related plant variety or species, or thede novo assembled genome sequence of one or more related plant varietiesor species. It is possible for example to detect polymorphisms for thecharacteristic of bolting by comparing the genotype and/or the sequenceof lettuce variety 45-21 RZ with the genotype and/or the sequence of oneor more reference plants. The reference plant(s) used for comparison mayfor example be, but is not limited to, any of the comparison varieties45-89 RZ. Elenas or Salinas. It is also possible for example, to detectpolymorphisms for the resistance to downy mildew (Bremia lactucae) racesBl:1 to Bl:28, CA-1-CA-VIII by comparing the genotype and/or thesequence of lettuce variety 45-21 RZ with the genotype and/or thesequence of one or more reference plants. The reference plant(s) usedfor comparison may for example be, but is not limited to, any of thecomparison varieties Elenas or Salinas.

The polymorphisms revealed by these techniques may be used to establishlinks between genotype and phenotype. The polymorphisms may thus be usedto predict or identify certain phenotypic characteristics, individuals,or even species. The polymorphisms are generally called markers. It iscommon practice for the skilled artisan to apply molecular DNAtechniques for generating polymorphisms and creating markers.

The polymorphisms of this invention may be provided in a variety ofmediums to facilitate use, e.g. a database or computer readable medium,which may also contain descriptive annotations in a form that allows askilled artisan to examine or query the polymorphisms and obtain usefulinformation.

As used herein “database” refers to any representation of retrievablecollected data including computer files such as text files, databasefiles, spreadsheet files and image files, printed tabulations andgraphical representations and combinations of digital and image datacollections. In a preferred aspect of the invention, “database” refersto a memory system that may store computer searchable information.

As used herein, “computer readable media” refers to any medium that maybe read and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM, DRAM, SRAM, SDRAM, ROM; and PROMs(EPROM, EEPROM, Flash EPROM), and hybrids of these categories such asmagnetic/optical storage media. A skilled artisan may readily appreciatehow any of the presently known computer readable mediums may be used tocreate a manufacture which may comprise computer readable medium havingrecorded thereon a polymorphism of the present invention.

As used herein, “recorded” refers to the result of a process for storinginformation in a retrievable database or computer readable medium. Forinstance, a skilled artisan may readily adopt any of the presently knownmethods for recording information on computer readable medium togenerate media which may comprise the polymorphisms of the presentinvention. A variety of data storage structures are available to askilled artisan for creating a computer readable medium where the choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats may be used to store the polymorphsims ofthe present invention on computer readable medium.

The present invention further provides systems, particularlycomputer-based systems, which contain the polymorphisms describedherein. Such systems are designed to identify the polymorphisms of thisinvention. As used herein, “a computer-based system” refers to thehardware, software and memory used to analyze the polymorphisms. Askilled artisan may readily appreciate that any one of the currentlyavailable computer-based system are suitable for use in the presentinvention.

Lettuce leaves are sold in packaged form, including without limitationas pre-packaged lettuce salad or as lettuce heads. Mention is made ofU.S. Pat. No. 5,523,136, incorporated herein by reference consistentwith the above INCORPORATION BY REFERENCE section, which providespackaging film, and packages from such packaging film, including suchpackaging containing leafy produce, and methods for making and usingsuch packaging film and packages, which are suitable for use with thelettuce leaves of the invention. Thus, the invention comprehends the useof and methods for making and using the leaves of the lettuce plant ofthe invention, as well as leaves of lettuce plants derived from theinvention.

The invention further relates to a container which may comprise one ormore plants of the invention, or one or more lettuce plants derived froma plant of the invention, in a growth substrate for harvest of leavesfrom the plant in a domestic environment. This way the consumer may pickvery fresh leaves for use in salads. More generally, the inventionincludes one or more plants of the invention or one or more plantsderived from lettuce of the invention, wherein the plant is in aready-to-harvest condition, including with the consumer picking his own,and further including a container which may comprise one or more ofthese plants.

The invention is further described by the following numbered paragraphs:

1. Lettuce plant exhibiting a combination of traits including resistanceto downy mildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I toCA-VIII, resistance to currant-lettuce aphid (Nasonovia ribisnigri)biotype Nr:0, very slow bolting, moderately to deeply incised margins ofthe mature leaves and a large head size, representative seed of whichhaving been deposited under NCIMB Accession No. 41901.

2. Lettuce plant of paragraph 1 wherein the plant exhibits a combinationof traits including resistance to downy mildew (Bremia lactucae) racesBl:1 to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves, a large head size andmoderate to slight blistering.

3. Lettuce plant designated 45-21 RZ, representative seed of whichhaving been deposited under NCIMB Accession No. 41901.

4. A seed of the plant of paragraph 1.

5. Parts of the plant of paragraph 1 or paragraph 2 or paragraph 3,wherein said parts of the plant are suitable for sexual reproduction.

6. Parts of the plant of paragraph 5, wherein said parts are selectedfrom the group consisting of microspores, pollen, ovaries, ovules,embryo sacs and egg cells.

7. Parts of the plant of paragraph 1 or paragraph 2 or paragraph 3,wherein said parts of the plant are suitable for vegetativereproduction.

8. Parts of paragraph 7, wherein said parts are selected from the groupconsisting of cuttings, roots, stems, cells and protoplasts.

9. A tissue culture of regenerable cells from the lettuce plant ofparagraph 1.

10. A tissue culture of paragraph 9, wherein said cells or protoplastsof the tissue culture which are derived from a tissue selected from thegroup consisting of leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.

11. Progeny of a lettuce plant of paragraph 1 or paragraph 2 orparagraph 3.

12. Progeny of paragraph 11, wherein said progeny is produced by sexualor vegetative reproduction of said lettuce plant, and wherein saidprogeny exhibits a combination of traits including resistance to downymildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size.

13. Progeny of a lettuce plant of paragraph 3, having all themorphological and physiological characteristics of the lettuce plant ofparagraph 3, representative seed of which having been deposited underNCIMB Accession No. 41901 wherein the morphological and physiologicalcharacteristics are as found in lettuce variety 45-21 RZ, representativeseed of which having been deposited under NCIMB Accession No. 41901.

14. Progeny of a lettuce plant of paragraph 1 or paragraph 2 orparagraph 3, representative seed of which having been deposited underNCIMB Accession No. 41901, and is modified in one or more othercharacteristics.

15. Progeny of paragraph 14, wherein the modification is effected bymutagenesis.

16. Progeny of paragraph 14, wherein the modification is effected bytransformation with a transgene.

17. A method of producing a hybrid lettuce seed comprising crossing afirst parent lettuce plant with a second parent lettuce plant andharvesting the resultant hybrid lettuce seed, wherein said first parentlettuce plant or said second parent lettuce plant is the lettuce plantof paragraph 1.

18. A hybrid lettuce plant produced by the method of paragraph 17.

19. A method of producing a lettuce cultivar containing a combination oftraits including resistance to downy mildew (Bremia lactucae) races Bl:1to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size,comprising: crossing a mother lettuce plant with a father lettuce plantto produce a hybrid seed; growing said hybrid seed to produce a hybridplant; selfing said hybrid plant to produce F2 progeny seed; growingsaid F2 progeny seed to produce F2-plants; selecting said F2-plants forexhibiting resistance to downy mildew (Bremia lactucae) races Bl:1 toBl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size, and,selfing said selected F2-plants to produce F3 progeny seed; growing saidF3 progeny seed to produce F3-plants; selecting F3-plants for exhibitingresistance to downy mildew (Bremia lactucae) races Bl:1 to Bl:28 andCA-I to CA-VIII, resistance to currant-lettuce aphid (Nasonoviaribisnigri) biotype Nr:0, very slow bolting, moderately to deeplyincised margins of the mature leaves and a large head size, optionallyfollowed by more selfing and selection steps.

20. A lettuce cultivar produced by the method of paragraph 19.

21. A method for producing lettuce leaves as a fresh vegetablecomprising packaging leaves of a plant of paragraph 1.

22. A method for producing lettuce leaves as a processed food comprisingprocessing leaves of a plant of paragraph 1.

23. One or more lettuce plants of paragraph 1, in a container, forharvest of leaves.

24. Lettuce plant having morphological and/or physiologicalcharacteristics of a lettuce plant, representative seed of which havingbeen deposited under NCIMB Accession No. 41901.

25. Lettuce plant of paragraph 23 having all the morphological andphysiological characteristics of the lettuce plant, representative seedof which having been deposited under NCIMB Accession No. 41901.

26. A method of introducing a desired trait into a plant of lettucevariety 45-21 RZ comprising: (a) crossing a plant of lettuce variety45-21 RZ, representative seed of which having been deposited under NCIMBAccession No. NCIMB 41901, with a second lettuce plant that comprisesthe desired trait to produce F1 progeny; (b) selecting an F1 progenythat comprises the desired trait; (c) crossing the selected F1 progenywith a plant of lettuce variety 45-21 RZ, to produce backcross progenyand (d) selecting backcross progeny comprising the desired trait and thephysiological and morphological characteristic of a plant of lettucevariety 45-21 RZ, when grown in the same environmental conditions.

27. The method of paragraph 26 further comprising (e) repeating steps(c) and (d) one or more times in succession to produce selected fourthor higher backcross progeny that comprise the desired trait and all ofthe physiological and morphological characteristics of a plant oflettuce variety 45-21 RZ, when grown in the same environmentalconditions.

28. A lettuce plant produced by the method of paragraph 26 or paragraph27.

29. A method for producing a seed of a 45-21 RZ-derived lettuce plantcomprising (a) crossing a plant of lettuce variety 45-21 RZ,representative seed of which having been deposited under NCIMB AccessionNo. NCIMB 41901, with a second lettuce plant, and (b) whereby seed of a45-21 RZ-derived lettuce plant forms.

30. The method of paragraph 29 further comprising (c) crossing a plantgrown from 45-21 RZ-derived lettuce seed with itself or with a secondlettuce plant to yield additional 45-21 RZ-derived lettuce seed, (d)growing the additional 45-21 RZ-derived lettuce seed of step (c) toyield additional 45-21 RZ-derived lettuce plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 45-21 RZ-derived lettuce plants.

31. The method of paragraph 30 further comprising selecting at steps b),d), and e), a 45-21 RZ-derived lettuce plant, exhibiting a combinationof traits including resistance to downy mildew (Bremia lactucae) racesBl:1 to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size.

32. Seed produced by the method of paragraphs 29 or 30 or 31.

33. A method of determining the genotype of a plant of lettuce variety45-21 RZ, representative seed of which has been deposited under NCIMBAccession No. NCIMB 41901, or a first generation progeny thereof,comprising obtaining a sample of nucleic acids from said plant andcomparing said nucleic acids to a sample of nucleic acids obtained froma reference plant, and detecting a plurality of polymorphisms betweenthe two nucleic acid samples, wherein the plurality of polymorphisms areindicative of and/or give rise to the expression of the morphologicaland physiological characteristics of lettuce variety 45-21 RZ.

34. The method of paragraph 33 additionally comprising the step ofstoring the results of detecting the plurality of polymorphisms on acomputer readable medium, or transmitting the results of detecting theplurality of polymorphisms.

35. The computer readable medium of paragraph 34.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention is not to belimited to particular details set forth in the above description as manyapparent variations thereof are possible without departing from thespirit or scope of the present invention.

What is claimed is:
 1. A lettuce plant exhibiting a combination oftraits including resistance to downy mildew (Bremia lactucae) races Bl:1to Bl:28 and CA-I to CA-VIII, resistance to currant-lettuce aphid(Nasonovia ribisnigri) biotype Nr:0, very slow bolting, moderately todeeply incised margins of the mature leaves and a large head size,representative seed of which having been deposited under NCIMB AccessionNo.
 41901. 2. The lettuce plant of claim 1 wherein the plant exhibits acombination of traits including resistance to downy mildew (Bremialactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves, alarge head size and moderate to slight blistering.
 3. A lettuce plantdesignated 45-21 RZ, representative seed of which having been depositedunder NCIMB Accession No.
 41901. 4. A seed of the plant of claim
 1. 5.Parts of the plant of claim 1, wherein said parts of the plant aresuitable for sexual reproduction.
 6. Parts of the plant as claimed inclaim 5, wherein said parts are selected from the group consisting ofmicrospores, pollen, ovaries, ovules, embryo sacs and egg cells. 7.Parts of the plant of claim 1, wherein said parts of the plant aresuitable for vegetative reproduction.
 8. Parts as claimed in claim 7,wherein said parts are selected from the group consisting of cuttings,roots, stems, cells and protoplasts.
 9. A tissue culture of regenerablecells from the lettuce plant of claim
 1. 10. The tissue culture asclaimed in claim 9, wherein said cells or protoplasts of the tissueculture which are derived from a tissue selected from the groupconsisting of leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.11. A progeny of a lettuce plant of claim
 1. 12. The progeny as claimedin claim 11, wherein said progeny is produced by sexual or vegetativereproduction of said lettuce plant, and wherein said progeny exhibits acombination of traits including resistance to downy mildew (Bremialactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size.
 13. A progeny of a lettuce plant of claim 3, having allthe morphological and physiological characteristics of the lettuce plantof claim 3, representative seed of which having been deposited underNCIMB Accession No. 41901 wherein the morphological and physiologicalcharacteristics are as found in lettuce variety 45-21 RZ, representativeseed of which having been deposited under NCIMB Accession No.
 41901. 14.A progeny of a lettuce plant of claim 1, representative seed of whichhaving been deposited under NCIMB Accession No. 41901, and is modifiedin one or more other characteristics.
 15. The progeny as claimed inclaim 14, wherein the modification is effected by mutagenesis.
 16. Theprogeny as claimed in claim 14, wherein the modification is effected bytransformation with a transgene.
 17. A method of producing a hybridlettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant hybrid lettuceseed, wherein said first parent lettuce plant or said second parentlettuce plant is the lettuce plant of claim
 1. 18. A hybrid lettuceplant produced by the method of claim
 17. 19. A method of producing alettuce cultivar containing a combination of traits including resistanceto downy mildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I toCA-VIII, resistance to currant-lettuce aphid (Nasonovia ribisnigri)biotype Nr:0, very slow bolting, moderately to deeply incised margins ofthe mature leaves and a large head size, comprising: crossing a motherlettuce plant with a father lettuce plant to produce a hybrid seed;growing said hybrid seed to produce a hybrid plant; selfing said hybridplant to produce F2 progeny seed; growing said F2 progeny seed toproduce F2-plants; selecting said F2-plants for exhibiting resistance todowny mildew (Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII,resistance to currant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0,very slow bolting, moderately to deeply incised margins of the matureleaves and a large head size, and, selfing said selected F2-plants toproduce F3 progeny seed; growing said F3 progeny seed to produceF3-plants; selecting F3-plants for exhibiting resistance to downy mildew(Bremia lactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size, optionally followed by more selfing and selectionsteps.
 20. A lettuce cultivar produced by the method of claim
 19. 21. Amethod for producing lettuce leaves as a fresh vegetable comprisingpackaging leaves of a plant of claim
 1. 22. A method for producinglettuce leaves as a processed food comprising processing leaves of aplant of claim
 1. 23. One or more lettuce plants of claim 1, in acontainer, for harvest of leaves.
 24. A lettuce plant havingmorphological and/or physiological characteristics of a lettuce plant,representative seed of which having been deposited under NCIMB AccessionNo.
 41901. 25. The lettuce plant of claim 23 having all themorphological and physiological characteristics of the lettuce plant,representative seed of which having been deposited under NCIMB AccessionNo.
 41901. 26. A method of introducing a desired trait into a plant oflettuce variety 45-21 RZ comprising: (a) crossing a plant of lettucevariety 45-21 RZ, representative seed of which having been depositedunder NCIMB Accession No. NCIMB 41901, with a second lettuce plant thatcomprises the desired trait to produce F1 progeny; (b) selecting an F1progeny that comprises the desired trait; (c) crossing the selected F1progeny with a plant of lettuce variety 45-21 RZ, to produce backcrossprogeny and (d) selecting backcross progeny comprising the desired traitand the physiological and morphological characteristic of a plant oflettuce variety 45-21 RZ, when grown in the same environmentalconditions.
 27. The method of claim 26 further comprising (e) repeatingsteps (c) and (d) one or more times in succession to produce selectedfourth or higher backcross progeny that comprise the desired trait andall of the physiological and morphological characteristics of a plant oflettuce variety 45-21 RZ, when grown in the same environmentalconditions.
 28. A lettuce plant produced by the method of claim
 26. 29.A method for producing a seed of a 45-21 RZ-derived lettuce plantcomprising (a) crossing a plant of lettuce variety 45-21 RZ,representative seed of which having been deposited under NCIMB AccessionNo. NCIMB 41901, with a second lettuce plant, and (b) whereby seed of a45-21 RZ-derived lettuce plant forms.
 30. The method of claim 29 furthercomprising (c) crossing a plant grown from 45-21 RZ-derived lettuce seedwith itself or with a second lettuce plant to yield additional 45-21RZ-derived lettuce seed, (d) growing the additional 45-21 RZ-derivedlettuce seed of step (c) to yield additional 45-21 RZ-derived lettuceplants, and (e) repeating the crossing and growing of steps (c) and (d)for an additional 3-10 generations to generate further 45-21 RZ-derivedlettuce plants.
 31. The method of claim 30 further comprising selectingat steps b), d), and e), a 45-21 RZ-derived lettuce plant, exhibiting acombination of traits including resistance to downy mildew (Bremialactucae) races Bl:1 to Bl:28 and CA-I to CA-VIII, resistance tocurrant-lettuce aphid (Nasonovia ribisnigri) biotype Nr:0, very slowbolting, moderately to deeply incised margins of the mature leaves and alarge head size.
 32. A seed produced by the method of claim
 29. 33. Amethod of determining the genotype of a plant of lettuce variety 45-21RZ, representative seed of which has been deposited under NCIMBAccession No. NCIMB 41901, or a first generation progeny thereof,comprising obtaining a sample of nucleic acids from said plant andcomparing said nucleic acids to a sample of nucleic acids obtained froma reference plant, and detecting a plurality of polymorphisms betweenthe two nucleic acid samples, wherein the plurality of polymorphisms areindicative of and/or give rise to the expression of the morphologicaland physiological characteristics of lettuce variety 45-21 RZ.
 34. Themethod of claim 33 additionally comprising the step of storing theresults of detecting the plurality of polymorphisms on a computerreadable medium, or transmitting the results of detecting the pluralityof polymorphisms.
 35. The computer readable medium of claim 34.